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Expression of the Grifola frondosa Trehalose Synthase Gene and Improvement of Drought-Tolerance in sugarcane （Saccharum officinarum L.）

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Journal of Integrative Plant Biology 2006年第4期48卷 453-459页

作者： Shu-Zhen Zhang Ben-Peng Yang Cui-Lian Feng Ru-Kai Chen Jing-Ping Luo Wen-Wei cai Fei-Hu Liu State Key Laboratory of tropical Crop biotechnology Chinese Academy of Tropical Agricultural Sciences Haikou 571101 China institute of tropical biotechnology Chinese Academy of Tropical Agricultural Sciences Haikou 571101 China Key Laboratory of Ecophysiology and Genetic Improvement for sugarcane Ministry of Agriculture Fuzhou 350002 China College of Life Sciences Yunnan University Kunming 650091 China

Trehalose Is a nonreduclng dlsaccharlde of glucose that functions as a protectant In the stabilization of blologlcal structures and enhances stress tolerance to abiotic stresses in organisms. We report here the expression of a Grlfola frondosa trehalose synthase （TSase） gene for Improving drought tolerance In sugarcane （Saccharum offlclnarum L.）. The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and transferred Into sugarcane by Agrobacterium tumefaciens EHA105. The transgenlc plants accumulated high levels of trehalose, up to 8.805-12.863 mg/g fresh weight, whereas It was present at undetectable level in nontransgenlc plants. It has been reported that transgenlc plants transformed with Escherlchla coil TPS （trehalose-6-phosphatesynthase） and/or TPP （trehalose-6-phosphate phosphatase） are severely stunted and have root morphologlc alterations. Interestingly, our transgenlc sugarcane plants had no obvious morphological changes and no growth Inhibition in the field. Trehalose accumulation in 35S-35S：TSase plants resulted In In- creased drought tolerance, as shown by the drought and the drought physiological Indexes, such as the rate of bound water/free water, plasma membrane permeability, malondlaldehyde content, chlorophyll a and b contents, and activity of SOD and POD of the excised leaves. These results suggest that transgenlc plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought.

关键词： drought tolerance Grifola frondosa Saccharum officinarum trehalose synthase gene.

Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii

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Journal of Zhejiang University-Science B(Biomedicine & biotechnology) 2013年第12期14卷 1121-1131页

作者： Xiao-dong DENG Jia-jia CAI Xiao-wen FEI Key Laboratory of tropical Crop biotechnology Ministry of Agriculture Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences School of Science Hainan Medical College

Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.

关键词： Phosphatidate phosphohydrolase 2 Triacylglycerol biosynthesis RNAi Chlamydomonas reinhardtii Nitrogen deprivation Over-expression

Biosensors: Types, features, and application in biomedicine

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Asian Pacific Journal of tropical Biomedicine 2022年第9期12卷 367-373页

作者： Elmira Karami Fatemeh Kazemi-Lomedasht biotechnology research center Biotechnology DepartmentPasteur Institute of IranTehranIran

Fast and precise diagnostic techniques are required for the treatment of many *** are one of the diagnostic devices that are applicable in biological and medical *** could be utilized to recognize biological molecules with high *** are consisted of different components and have different *** type of biosensor is used in a particular field according to its specific *** are a novel class of antibodies with small size,high affinity,and specificity to their *** unique properties of nanobodies make them appropriate tools for diagnostic *** this paper,we review biosensors,and their features and roles in ***/nanobody-based biosensors are also specifically discussed.

关键词： Biosensor Nanobody-based biosensor Antibodybased biosensor Biomedicine biotechnology

Characterization of a Novel β-thioglucosidase CpTGG1 in Carica papaya and its Substrate-dependent and Ascorbic Acid-independent O-β-glucosidase Activity

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Journal of Integrative Plant Biology 2010年第10期52卷 879-890页

作者： Han Nong Jia-Ming Zhang Ding-Qin Li Meng Wang Xue-Piao Sun Yun Judy Zhu Johan Meijer Qin-Huang Wang National center for tropical Crops Engineering and Technology research Spice and Beverage Crops Research Institute ChineseAcademy of Tropical Agricultural Sciences (CATAS) Wannin 571533 China MOA Key biotechnology Laboratory for tropical Crops Institute of Tropical Bioscience and Biotechnology CATAS Haikou 571101 China College of Agriculture Hainan University H'aikou 571101 China institute of biotechnology Yunnan Acaclemy of Agricultural Sciences Kunming 650205 China Department of Plant Biology and Forest Genetics BioCenter Swedish University of Agricultural Sciences SE-750 07 Uppsala Sweden

Plant thioglucosidases are the only known S-glycosidases in the large superfamily of glycosidases. These enzymes evolved more recently and are distributed mainly in Brassicales. Thioglucosidase research has focused mainly on the cruciferous crops due to their economic importance and cancer preventive benefits. In this study, we cloned a novel myrosinase gene, CpTGG1, from Carica papaya Linnaeus. and showed that it was expressed in the aboveground tissues in planta. The recombinant CpTGG1 expressed in Pichia pastoris catalyzed the hydrolysis of both sinigrin and glucotropaeolin （the only thioglucoside present in papaya）, showing that CpTGG1 was indeed a functional myrosinase gene. Sequence alignment analysis indicated that CpTGG1 contained all the motifs conserved in functional myrosinases from crucifers, except for two aglycon-binding motifs, suggesting substrate priority varia- tion of the non-cruciferous myrosinases. Using sinigrin as substrate, the apparent Km and Vmax values of recombinant CpTGG1 were 2.82 mM and 59.9 μmoI min-1 mg protein-1, respectively. The KcatlKm value was 23 s-1 mM-1, O-β-glucosidase activity towards a variety of substrates were tested, CpTGG1 displayed substrate-dependent and ascorbic acid-independent O-β-glucosidase activity towards 2-nitrophenyl-β- D-glucopyranoside and 4-nitrophenyl-β-D-glucopyranoside, but was inactive towards glucovanillin and n-octyl-β-D-glucopyranoside. Phylogenetic analysis indicated CpTGG1 belongs to the MYR II subfamily of myrosinases.

关键词： GLUCOSIDASES PLANT enzymes PAPAYA VITAMIN C PLANT gene expression CANCER prevention PLANT phylogeny PLANT cells & tissues

Cloning and Analysis of Chitinase Gene in Bacillus subtilis HAS

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Plant Diseases and Pests 2019年第2期10卷 4-6页

作者： Xiong Guoru Zhao Gengfeng Cai Wenwei Wang Jungang Yang Benpeng Zhang Shuzhen institute of tropical biotechnology/sugarcane research center CATAS Department of Biochemistry Hainan Medical College

[Objective] The paper was to study the possible mechanism of chitinase gene. [Method] The chitinase gene with inhibitory effect against a variety of pathogenic fungi was cloned and analyzed in this study. [Result] The genome of HAS strain contained an ORF of 834 bp. The homology of coding protein corresponding to ORF with chitosanase [Bacillus subtilis subsp. subtilis str. 168](ref|NP_390566.1) was the highest of 98%(272/277).[Conclusion]Through the analysis of amino acid sequence and secondary structure of the protease, tertiary structure prediction and analysis, HAS strain may play a pivotal role in controlling Sporisorium scitaminea Syd.

关键词： Bacillus subtilis HAS Sporisorium scitaminea Syd. Antifungal protein Chitinase gene

Annotation and validation of genes involved in photosynthesis and starch synthesis from a Manihot full-length cDNA library

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Frontiers of Agricultural Science and Engineering 2016年第4期3卷 308-320页

作者： Yang ZHANG Xin CHEN Haiyan WANG Zhiqiang XIA Peng LING Wenquan WANG institute of tropical Bioscience and biotechnology Chinese Academy of Tropical Agricultural Sciences Department of Horticultural Sciences Citrus Research and Education Center University of Florida

A full-length c DNA library from leaf and root tissues of cassava(Manihot esculenta) Arg7 and one accession of its wild ancestor W14(M. esculenta ***) has been constructed. The library is comprised of four sub-libraries, containing 32640 recombinant clones, 6028 c DNA clones from their 5′ ends, and 128 clones from the 3′ ends were sequenced. In total, 5013high-quality expressed sequence tags(ESTs) and 1259 unigenes were obtained. Of these, 746 unigenes were identified by their sequence homologies to ESTs from model plants, and 323 unigenes were mapped onto 114 different KEGG pathways. From these, 24 differentially expressed genes involved in starch metabolism and photosynthesis were identified and five of them were selected to compare their expression level between Arg7 and W14. Notably, Arg7 has a higher net photosynthesis rate in leaves, higher ribulose-1,5-bisphosphate carboxylase oxygenase activities in leaves, and higher AGPase activity in roots. This resource is the first EST collection from wild cassava and should be of value for gene discovery, genome annotation and studies of Manihot evolution.

关键词： Manihot esculenta expressed sequence tag unigene pathway expression pattern

Complementation and Expression Analysis of SoRablA and SoRab2A in sugarcane Demonstrates Their Functional Diversification

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Journal of Integrative Plant Biology 2006年第12期48卷 1450-1457页

作者： Jia-Ming Zhang Anne W Sylvester Ding-Qin Li Xue-Piao Sun State Key biotechnology Laboratory for tropical Crops Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences Haikou 571101 China Department of Botany University of Wyoming Laramie Wyoming 82071 USA

Mammalian and plant Rabl and Rab2 are small GTPases that regulate vesicle trafficking in the endoplasmic reticulum （ER） to Golgi compartments. Little is known about their functional diversification or potential interaction. We cloned sugarcane （Saccharum officinarum L.） Rab1A and Rab2A genes and studied their functional differences by expression and complementation experiments. We found differential expression of the two genes during sugarcane leaf development： SoRab2A expression declined from the dividing base to the maturing tip of the growing leaves, whereas SoRab1A was constitutively expressed, suggesting that SoRab2A is required for cell division and expansion and SoRablA is required for cells at all developmental stages. We used a yeast temperature sensitive ypt1-A 136D mutant strain to further investigate these shared and unique functions. Yptl is a small GTPase that regulates vesicle transport in the same cellular location as Rabl and Rab2. Neither SoRab1A nor SoRab2A alone could restore the growth of the mutant at restrictive temperatures when SoRab1A and SoRab2A were transformed separately. However, SoRab1A transformants maintained normal morphology and viability at non-permissive temperature, and resumed growth when returned to permissive temperature, whereas SoRab2A transformants died at non-permissive temperature, suggesting that SoRablA function is required for a cell＇s viability. Mutant growth was fully restored when SoRab1A and SoRab2A were co-transformed, indicating that SoRablA and SoRab2A complement each other and they both are needed to restore the function of ypt1-A136D. These results demonstrate that SoRab1A and SoRab2A serve distinct but overlapping functions, mostly by regulating the transportation of different sets of proteins.

关键词： complementation co-transformation functional diversification Rab GTPase sugarcane temperature sensitivemutant

HPLC-ESI-MS Analysis of Flavonoids Obtained from Tissue Culture of Dracaena cambodiana

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Chemical research in Chinese Universities 2015年第1期31卷 38-43页

作者： WANG Hui WANG Jiayuan WU Xiaopeng SHEN Haiyan LUO Ying DAI Haofu MEI Wenli Hainan Key Laboratory for research and Development of Natural Products from Li Folk Medicine Key Laboratory of tropical Crop biotechnology Ministry of Agriculture Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences Haikou 571101 P. R. China

Dragon＇s blood is a traditional medicine and used in many countries with different cultures because of its various therapeutic properties. The main bioactive constituents of dragon＇s blood are flavonoids, which exhibit various pharmaceutical activities, such as haemostatic, analgesic, anticoagulant, and antimicrobial activities and so on, and have attracted the attention of researchers on the development of new drugs. However, the formation process of dragon＇s blood in nature is very slow, which cannot meet the demand of pharmaceutical uses. Dracaena cambodiana （D. cambodiana） is one of the resource plants of dragon＇s blood na, 6-benzylaminopurine was added in the medium, then red During the course of tissue culture of D. cambodiasecretion was discovered in the cultured medium. Twelve compounds（1--12）, including 11 flavonoids, were determined via the analysis of constituents of the cultured medium by high performance liquid chromatography-electrospray ionization-mass spectrometry（HPLC-ESI-MS） and the results were compared with that of the standards isolated from dragon＇s blood. Among compounds 1-12, 4,4＇- dihydroxy-2-methoxydihydrochalcone（5）, 4,4＇-dihydroxy-2,6-dimethoxydihydrochalcone（6） and （2S）-7,3＇-dihydroxy- 4＇-methoxyflavane（9） were major compounds of the red secretion, with contents of 15.70, 7.10 and 57.23 mg/L, respectively. Therefore, it is promising that flavonoids from dragon＇s blood can be obtained from the tissue culture of its resource plants for the purpose of drug development.

关键词： Flavonoid Dragon's blood High performance liquid chromatography-electrospray ionization-mass spectrometry(HPLC-ESI-MS) Dracaena cambodiana Tissue culture

Cloning and Analysis of TasA Gene in Bacillus subtilis HAS

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Agricultural biotechnology 2017年第2期6卷 48-50,54页

作者： Guoruo XIONG Gengfeng ZHAO Wenwei CAI Suran WU Benpeng YANG Shuzhen ZHANG institute of tropical biotechnology/sugarcane research center Chinese Academy of Tropical Agricultural Sciences Department of Biochemistry Hainan Medical College

In order to clarify the mechanism for the inhibitory action of the Bacillus subtilis strain HAS on Sporisorium scitamineum （ Syd. ）, which can cause sugarcane smut, the full-length TasA gene which encodes a protein with broad-spectrum antimicrobial activity, was PCR-amplified from HAS, and cloned into pMD18-T vector. Sequence analysis indicated that the full-length TasA cloned from HAS consisted of 786 nucleotides, and shared 99% homology in nucleotide sequence with the TasA gene sequence published in Genbank （AJ871386.1）. It was predicted that the molecular weight of TasA protein was about 28 kD. Base transitions or transversions ~curred at positions 104, 164, 169,250, 399,623 and 627, at the 2nd, 2nd, 3rd , 1st , 3rd, 2nd and 3rd bases of TasA codons. The mutations in the seven bases may cause the missense mutations of the polypeptide chain. Compared with the amino acid sequences of the TasA protein encoded by Bacillus aubtilis subsp, subtilis str. 168, mutations in two amino acids at positions 150 and 209 of the protein encoded by the cloned TasA gene were found, and as a result, an ala- nine was replaced with a threonine.

关键词： Bacillus subtilis HAS mad a glutamic acid was replaced with an asparagine. Sporisorium scitamineum ( Syd. ) Antimicrobial protein TasA